Hormone-mediated modulation of gene activation or repression through transcription factors is central to all organisms. Auxin Response Factor (ARF) transcription factors are critical modulators of plant growth and provide an ideal model for exploring hormone control of gene activation and repression. We have recently identified protein multimerization and proteasomal degradation as two previously unknown mechanisms that regulate ARF activity. The long-term goal of this research project is to determine the importance of these ARF regulatory mechanisms in Arabidopsis thaliana transcriptional control. Elucidating the molecular mechanism of ARF regulation likely will uncover control processes common to other transcription factors. A repression-derepression paradigm regulates ARF activity. Under low concentrations of the hormone auxin, ARF transcriptional activity is repressed by Aux/IAA repressor proteins. When auxin concentrations increase, a co-receptor complex, comprised of an F-box protein (TIR1) and an Aux/IAA repressor protein, directly binds auxin. The F-box protein participates in a Skp1-Cullin-F-box (SCF) E3 ubiquitin ligase, which targets the Aux/IAA protein for degradation. This degradation event relieves ARF repression, thereby allowing auxin-regulated gene transcription. Although this molecular model of repression and derepression for auxin activity appears relatively simple, our recent preliminary data suggest several exciting new control components and posttranslational modifiers influence ARF transcriptional activity and protein accumulation. This project aims to elucidate auxin signaling molecular mechanisms by identifying regulators of ARF protein activity and accumulation. To achieve this goal, we will use a combination of biochemical, biophysical, cell biology, synthetic biology, molecular and genetic techniques to gain insight into factors that influence ARF activity. The first aim is to understand the role of protein multimerization in the regulation of ARF transcriptional activity. Studies in both a synthetic yeast auxin response system and in planta will be used to test aspects of this aim, which includes functional assays and 3C analysis. The second aim is understand the role of multimerization in the regulation of ARF localization. We will determine whether ARF posttranslational modification affects ARF cellular localization. Our third aim is to establish roles for ARF proteasome-dependent degradation in regulating auxin response and plant development. We will use a variety of genetic, biochemical, and cell biology techniques to understand the biological and developmental roles for regulated ARF stability. The proposed research is innovative because our approaches focus strongly on the molecular understanding of ARF regulation, guided by our recent structural data on ARF7 and it has the potential to dramatically alter the auxin signaling model. The proposed research is significant because it is expected to advance and expand understanding of transcription factor regulation, using ARF factors as a model.